The aim of this research application is to develop a paradigm shift in diagnostic testing for a globally important, yet poorly diagnosed, healthcare-associated infectious disease, namely Clostridium difficile infection (CDI). CDI results from infection of the bowel by C. difficile, a Gram-positive, spore-forming, obligate anaerobic bacterium. It can cause (diarrheal) illness ranging from moderate diarrhea to severe colitis (toxic megacolon) with a high risk of death. For the last two decades CDI has re-emerged in healthcare facilities with nearly a 10- fold increase in mortality and significant excess healthcar cost. Current CDI diagnostics is limited to detection of the organism and/or its toxin product(s) i conjunction with clinical symptoms, and does not differentiate infected from simply colonized patients, thus leading to inaccurate diagnosis as well as antibiotic mis/overuse. To overcome these limitations and resolve uncertainty of CDI diagnosis, a ground-breaking concept for CDI diagnosis will be developed and evaluated during this research project. The project rationale is that C. difficile toxin B mechanism of action causes cytoskeletal disruption of colonic epithelial cells with release of non- muscle tropomyosin (Tm). Our hypothesis is that increased levels of Tm, a major protein in colonic epithelial cytoskeleton, in the patient's stool indicates a specific host response to CDI and can be used as a diagnostic target signifying active infection from C. difficile. The application objective is to develop and evaluate Tm detection in stool specimens as a biomarker for clinically significant manifestations of CDI. To accomplish it, we will pursue two specific aims: 1) Determine the feasibility of clinically appropriate fecal Tm detection. This aim will be accomplished by performing multiple tasks - generation of Tm specific antibodies, optimization of Tm recovery method from stool specimens, Tm detection protocol development and optimization utilizing controls mimicking CDI negative and positive specimens, and testing for potential interferences. 2) Evaluate fecal Tm as a CDI specific host response marker on specimens obtained from clinically diverse patient populations. For that, CDI positive and negative clinical specimens will be utilized for Tm detection and data comparison to clinical and microbiological records of the same specimens. The records are available due to access and use of remnant samples from the clinical microbiology laboratory. The specificity of CDI diagnostics will be validated on stool specimens from patients with diarrhea due to other enteric bacterial pathogens (E. coli, Salmonella, Shigella spp, and Campylobacter), parasites, antibiotic-associated diarrhea (not due to infection), and bowel disorders like inflammatory bowel disease. This proposed concept has great potential to allow development of a novel diagnostic application for CDI. It will serve as an example for the next generation of infectious disease diagnostics measuring host response instead of detecting pathogens or their products. Our long term goal is to improve patient care by delivering a host response CDI test ready for development with an industry partner, such as TechLab(r), Inc., as a commercial assay that can undergo FDA clearance trials.